The long-term goal of our proposed study is to identify novel epigenetic mechanisms for regulating important biological process. Investigating epigenetic silencing complexes in controlling mouse embryonic stem cells (ESCs) fate provides an ideal platform for our investigation. Our recent work has identified the novel role of Rif1 gene as an epigenetic silencing factor for 2-Cell embryo genes and trophectoderm genes ESCs. This repression is critical for cell fate maintenance in ESCs. Previous studies showed that a rare family of endogenous retrotransposon (<0.05%), MERV-L, serves as promoters for 2-Cell embryo gene expression but little is known about the regulatory mechanisms. In addition, trophectoderm genes need to be silenced in order to maintain the commitment of ESCs to embryonic tissues but the molecular underpinning remains elusive. Besides these intriguing issues, a longstanding interest in the field is to develop simple and reliable approaches to study epigenetic factors in a locus-specific manner. Our most recent preliminary studies have identified a novel epigenetic silencing complex in ESCs, Rif1 epigenetic silencing complex (RESC). Our studies also led to unexpected mechanisms for RESC-mediated silencing. First, we demonstrated that interstitial telomeric sequences (telomere-like sequences located in intrachromosomal sites), which are strongly enriched at flanking regions of MERV-L, play a critical role in RESC-mediated silencing of 2-Cell embryo genes. Second, Rif1's interaction with pluripotency transcription factors is critical for repressing trophectoderm genes, which don't have MERV-L as promoters. Based on these findings, we hypothesize that RESC utilizes distinct mechanisms for targeting different genes. Furthermore, we have been developing new approaches that are based on the modified CRISPR/Cas9 systems to study epigenetic silencing in a locus specific manner. These approaches can tether epigenetic factors to target genes (CRISPR tethering) or pull out factors from specific loci by biochemistry means (CRISPR pull-out), respectively. In this proposal, we will fully investigate the mechanisms underlying RESC- mediated silencing and fully develop CRISPR-based approaches for studying epigenetic silencing. The significance and innovation of this study lie in several fronts. First, the outcomes will reveal novel mechanisms for recruiting epigenetic factors as well as cell fate decisions in ESC. Second, this work will add an interesting twist to the classic position variegation effects (o telomere position effect), i.e. the role of interstitial telomeric sequences in the epigenetic silencing of early embryonic genes, which has never been reported in mammalian cells. Third, the new methodologies under development will provide useful tools for studying transcriptional regulation and epigenetics in a locus specific manner.